This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We focus in our laboratory on developmental interactions between growing axons and glial cells with the goal of understanding the role of glia in axon navigation toward their targets in the CNS. We would like to characterize axonal growth cone behavior during the process of axon sorting in a region of the olfactory pathway that is populated by glial cells and in which olfactory receptor axons sort from one other in accord with their target region in the olfactory lobe. We have developed an organotypic preparation that comprises the entire primary olfactory pathway of the moth Manduca sexta and maintains the 3D environment through which the receptor axons normally extend to allow us to analyze the behavior of axonal growth cones as they extend near or in contact with the glia. These data will serve as the foundation for experiments in which we follow growth cone behavior when glial number is reduced or when the molecular environment in altered. Because our current time-lapse imaging of growing axons using standard confocal microscopy is marked by challenges of photo-bleaching, photo-toxicity and limited depth resolution, we need expert assistance in imaging our preparation with a multi-photon system, both to acquire preliminary data and to optimize our protocols for working with such a system.